In 2010, we made progress in the following projects: TCR signaling in response to partial agonists, Fos induction in T cells, quantitative analysis of cytokine receptor expression levels on T cells and developing tools to study gene expression and tune diffusion coefficients of proteins in glass supported lipid bilayers. We have an interest in understanding the signaling pathways activated downstream of TCR in response to low potency MHC-peptide complexes. Based on our previous work we have observed that there are a class of TCR ligands that generate Ras signals in the absence of calcium signals. Our hypothesis to explain this observation is that these Ras signals are generated by recruitment of Grb2-Sos module to the TCR complex in microclusters without recruitment or phophorylation of LAT. We have now made several observations to support our hypothesis. We have visualized the recruitment of GFP tagged LAT, Zap70 and Grb2 transfected in in-vitro activated AND TCR transgenic T cells using Total Internal Reflection Fluorescence microscopy (TIRFM) of cells interacting with glass supported lipid bilayers containing lipid anchored peptid-MHC complexes, ICAM-1 and CD80. We setup a two channel simultaneous imaging system for these experiments so that there is no delay between the TCR and GFP images. We find that the low potency ligands do not recruit LAT to the TCR microclusters and only poorly recruit Zap-70. On the other hand we see a specific recruitment of Grb2 to TCR microclusters generated in response to the low potency ligands over background. We have written a software program that in an automated way extracts quantitative information about the no. of microclusters and the relative amounts of fluorescence associated with it. Since Grb2 can recruit Sos which then acts as a Guanine nucleotide exchange factor for Ras, this explains the generation of Ras signals in the absence of Calcium signals in this setting. We want to understand the relationship between TCR engagement events at the cell surface and activation of transcription factors in the nucleus. For these experiments we have obtained transgenic mice that express a fusion protein between the transcription factor Fos and GFP under the control of the Fos promoter. Fos is not expressed in T cells in the basal state and is induced upon signaling. We have crossed these mice to AND TCR transgenic mice and have begun to study the induction of Fos using single cell imaging in response to ligands of varying strengths. We find that Fos is induced within 20 minutes of interaction with antigen and continues to accumulate for three hours after which the expression plateaus presumably due to degradation of Fos. Given that it takes about 6-7 minutes for GFP to mature, it is likely that the kinetics of Fos induction are even faster. Fos expression is controlled by MAPK activity in various cell types. Active Erk is known to phosphorylate members of the ternary complex factors, Elk-1, Sap-1 and Net which cause the transcription of Fos. MAPK signaling occurs downstream of TCR, however, how strength of TCR signaling affects the efficacy of this signaling pathway is not known. We find that Fos induction follows distinct kinetics depending on whether the TCR signals cause calcium signaling or not. The kinetics of Fos induction is more rapid in response to ligands that cause calcium signaling. We dont think that this has anything to do with calcium signaling but more to do with how Ras is activated in the two scenarios. Expectedly we find that Fos induction can be blocked by Erk and p38 inhibitors. These experiments will help us understand the hierarchy of MAPK signaling in T cells. We have also generated constructs of NFAT and p65 fused to tag-RFP (a red fluorescent protein) which we are simultaneously expressing in these cells so that we can follow the dynamics of two transcription factors at the same time. We are using endogenous promoters to express these transcription factors in T cells so that they are expressed at physiological levels and we are learning a lot regarding the requirements of expression of p65 in T cells. These reagents will allow us to address multiple questions concerning the single cell dynamics of transcription factors which cannot be addressed using biochemical analysis. How multiple subunit containing receptors such as antigen receptors whose ligand binding subunit is different from the signal transducing subunit, communicate information from ligand binding to signal transduction is not known. Fluorescence Resonance Energy Transfer (FRET) is a fluorescence phenomenon in which fluorophores transfer energy among themselves only when they are in molecular proximity of each other. Hence if FRET measurements are done at high time resolution, it offers the possibility of studying the dynamics of individual subunits within a receptor complex. The T cell receptor is very complex as it contains multiple subunits, and hence to establish a proof of principle approach we wanted to study a more simplified system which is equally relevant to the immune system. So we decided to investigate cytokine receptors belonging to the common gamma chain family. Before setting out to do the experiment it was important first to determine the physiological amounts of subunit of this family (IL2Ralpha, IL2Rbeta, IL15Ralpha, IL7Ralpha, IL4Ralpha, IL21Ralpha and the gamma chain) on the surface of naive and activated T cells. Unexpectedly we find that the levels of gamma chain are limiting when compared to the sum of all the other chains that it can pair up with. We are intrigued by this result and are exploring the significance of this in terms of signaling via these receptors. For example this may mean that under competing levels of certain cytokines one could potentially observe competition. We indeed find that when we have saturating amounts of IL-7 the cells are unable to signal via the IL-4 receptor. When we combine these experiments with FRET measurements we will be able to answer several questions: Is association between subunits of cytokine receptor governed by the cytokine driven affinity conversion model? What are the mechanisms of triggering of cytokine receptors and what is the speed with which they can be triggered? Diffusion of MHC molecules in the plasma membrane of antigen presenting cells affects how they trigger TCRs. To study this phenomenon using glass supported bilayers, we are trying to develop a system where we can tune the diffusion coefficient of molecules in the bilayer. If transmembrane anchored molecules are incorporated in bilayers, their cytoplasmic tail interacts with glass and gets stuck and hence they don't diffuse. We are exploring the possibility that if we incorporate a cytoplasmic tail deleted protein, then it would have reduced interaction with glass and hence may diffuse slower than a lipid anchored protein. We could then modulate the thickness of the bilayer using different lipids and thereby tune the diffusion coefficients of the incorporated proteins. We first made several truncated CD80 molecules, however, we found that they ended up becoming GPI anchored when expressed in CHO cells. Using bioinformatics software that would predict whether a protein sequence is likely to be GPI linked or not we found that the transmembrane domain of CD28 when truncated is not likely to be GPI-anchored. Using this we have generated several CD80 molecules containing the TM domain of CD28. We will first test if these are GPI anchored or not and then purify them and test their mobility in glass supported lipid bilayers.